CN102164675B

CN102164675B - Exchangeable carriers pre-loaded with reagent depots for digital microfluidics

Info

Publication number: CN102164675B
Application number: CN200980139397.XA
Authority: CN
Inventors: A·R·惠勒; I·巴布洛维科-纳德; H·杨; M·阿布德尔盖瓦德
Original assignee: University of Toronto; Tecan Trading AG
Current assignee: University of Toronto
Priority date: 2008-10-01
Filing date: 2009-09-30
Publication date: 2014-11-12
Anticipated expiration: 2029-09-30
Also published as: HK1158134A1; CN102164675A; EP2334434A1; CA2739000C; AU2009299892B2; US8187864B2; US20110240471A1; EP2334434B1; US8993348B2; AU2009299892A1; CA2739000A1; WO2010037763A1; US20100081578A1

Abstract

The present invention provides exchangeable, reagent pre-loaded carriers (10), preferably in the form of plastic sheets, which can be temporarily applied to an electrode array (16) on a digital microfluidic (DMF) device (14). The carrier (10) facilitates virtually un-limited re-use of the DMF devices (14) avoiding cross- contamination on the electrode array (16) itself, as well as enabling rapid exchange of pre-loaded reagents (12) while bridging the world-to-chip interface of DMF devices (14). The present invention allows for the transformation of DMF into a versatile platform for lab-on-a-chip applications.

Description

Be used for the replaceable carrier of the prepackage reagent storage bin of digital micro-fluid

Related application

Present patent application requires the U.S. Patent application the 12/285th of submitting on October 1st, 2008, the priority of No. 326.

Technical field

The present invention relates to the replaceable carrier for the pre-installed reagents of digital micro-fluid, more specifically, the present invention relates to removable plastic tab, on these thin slices, reagent has the replaceable carrier being placed on previously selected position as digital micro-fluid (DMF) device tactfully.

Background of invention

Microfluid relates to accurate control and the operation of the liquid that only has geometrically micro-volume (being generally microlitre).Because rapid kinetics and automatization level, microfluid is likely converted into conventional biologic test test reliably fast outside laboratory.There is recently the new example of a microminiaturized biologic test, be called " numeral " (or droplet base) microfluid.The surface that digital micro-fluid (DMF) relies on patterned electrodes operates discrete fluid drop, for example can be referring to US 7,147,763; US 4,636,785; US 5,486,337; US 6,911,132; US 6,565,727; US7,255,780; JP 10-267801; Or article " Electrowetting and electrowetting-on-dielectric for microscale liquid handling " the Sensors & Actuators 95:259-268 that delivered in 2002 of the people such as Lee; Article " Electrowetting-based actuation of liquid droplets for microfluidic applications " the Applied Physics Letters 77:1725-1726 that the people such as Pollack delivered in 2000; And Washizu, article " Electrostatic actuation of liquid droplets for microreactor applications " the IEEE Transactions on Industry Applications 34:732-737 that the people such as M. delivered in 1998.This technology type is similar to and in test tube, processes sample, is applicable to very much array biologic test, and in array biologic test, people can merge fluid drop or mix and carry out various biochemical reactions.The more important thing is, the array geometry of digital micro-fluid seems to be applicable to very much large-scale, parallel multiple analysis in itself.In fact, this new technology is applied aspect a lot, comprises taking cell as basic test, enzyme test, proteinogram and polymerase chain reaction.

Unfortunately, biofouling is connected these two major limitation and has restricted the range of application of digital micro-fluid with interface.Previous restriction is that biofouling has an adverse side effect for surface area and the V/V of whole micro-be all harmful to-Gao of small-scale analysis, is exactly analyte is adsorbed to surface of solids speed increase from solution.The applicant and other people have designed various strategies and have limited the biofouling degree of digital micro-fluid, but this problem becomes an obstacle all the time, stops the extensive use of this technology.

Second restriction of digital micro-fluid (with all microfluid systems) is that " extraneous and chip (world-to-chip) " interface-the most difficult is that reagent and sample are delivered to these systems, does not abandon again the intrinsic advantage that rapid analysis and low reagent consume.For being basic method taking microchannel, the scheme addressing this problem is to use pre-installed reagents.These methods generally comprise two steps:

(1) reagent is stored in to (or being stored in replaceable cartridge) in microchannel, and

(2), after, use rapidly reagent to carry out desirable test/experiment.

Two kinds of strategies of micro channel systems employing-in the first strategy, reagent is stored into droplet solution, with vent plug (article " Reagent-loaded cartridges for valveless and automated fluid delivery in microfluidic devices " the Analytical Chemistry 77:64-71 delivering in 2005 referring to people such as Linder) or by immiscible fluid (article " Microgram-scale testing of reaction conditions in solution using nanoliter plugs in microfluidics with detection by MALDI-MS " the Journal of the American Chemical Society 128:2518-2519 delivering in 2006 referring to people such as Hatakeyama, and article " A microfluidic approach for screening submicroliter volumes against multiple reagents by using preformed arrays of nanoliter plugs in athree-phase liquid/liquid/gas flow " the Angewandte Chemie-Intemational Edition 44:2520-2523 that delivered in 2005 of the people such as Zheng) drop is isolated mutually until use.In the second strategy, reagent is stored in passage with solid phase, then in the time testing, is reassembled as solution (article " The micro active project:Automatic detection of disease-related molecular cell activity " the Proceedings of SPIE-Int.Soc.Opt.Eng. that the people such as Furuberg delivered in 2007; Article " Controlled microfluidic reconstitution of functional protein from an anhydrous storage depot " the Lab on a Chip 4:78-82 that the people such as Garcia delivered in 2004; And article " Autonomous capillary system for one-step immunoassays " the Biomedical Microdevices that delivered in 2008 of the people such as Zimmermann).In microfluidic device, pre-installed reagents is to be applied in very many-sided a kind of strategy.But, up to now, in digital micro-fluid, still find no similar technology.

In order to solve the double challenge of in digital micro-fluid non-specific adsorption and World-to-chip interfacing, the applicant has researched and developed a kind of New Policy, cap based on removable (article " Low-cost, rapid-prototyping of digital microfluidics devices " the Microfluidics and Nanofluidics 4:349-355 delivering in 2008 referring to Abdelgawad and Wheeler; Article " Direct handwriting manipulation of droplets by self-aligned mirror-EWOD across adielectric sheet " the Proceedings of Mems:19th IEEE International Conference on Micro Electro Mechanical Systems that Chuang and Fan delivered in 2006, Technical Digest:538-541; And article " Two-dimensional electrostatic actuation of droplets using a single electrode panel and development of disposable plastic film card " the Sensors and Actuatorsa-Physical 136:358-366 that delivered in 2007 of the people such as Lebrasseur).After each experiment, change film, but reuse the maincenter structure of device.This prevents the cross pollution producing between replicate analysis effectively, may the more important thing is, as useful medium, reagent is introduced to microfluidic device.

Learn a kind of laser emission desorption apparatus for the formal operations liquid sample with indivedual drops from US 2008/0156983A1.Disclose the carrier that is preinstalled with reagent using with digital micro-fluid device.Described prepackage carrier has and is positioned at the locational one or more reagent storage bin of one or more pre-selected, and comprises electric insulation layer and hydrophobic surface.Described digital micro-fluid device comprises by dispersive electrode arranges the electrod-array and the electrode controller that form, and described electrode controller can optionally start and stop described dispersive electrode drop is moved on described hydrophobic surface to the described one or more pre-selected position on described prepackage carrier.Such drop is further directed on the specific pad of described digital micro-fluid device, can carry out MALDI (matrix-assisted laser desorption/ionization) and analyze on described specific pad.

Summary of the invention

For the principle that uses single battery lead plate and disposable plastic lid is described, the applicant is contained in dry enzyme spot on vinyl cover in advance, for later in proteolytic digestion test.Find, the reagent installing stores one month and still has above activity in household freezer.As the pioneer of this class technology, the applicant thinks that the present invention has represented that digital micro-fluid strides forward essential step, makes it become the fluid treatment platform that makes us attraction in various application.According to the present invention, even use two plates designs (have or without double electrode plate) to be also applicable to the carrier of pre-installed reagents.

The invention provides the removable such as plastic tab of disposable carrier of pre-installed reagents.This new method relates on the digital micro-fluid device that has connected prepackage carrier and operates reagent and sample.After analysis completes, have if required removable, analyze thin slice, in connection, after new prepackage thin slice, can reuse original device and start another test.

The removable disposable plastic thin slice of these pre-installed reagents contributes to use digital micro-fluid device to carry out rapid batch test, can not produce the problem of each test bay cross pollution.In addition, kit device disclosed herein and method contribute to use reagent storage bin.For example, inventor has manufactured the thin slice with the dry enzyme spot of prepackage, and described enzyme is generally applied in proteomics test, for example trypsase or alpha-chymotrypsin.After having digested model substrates ubiqutin, assess the thin slice that contains product with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).The present invention particularly advantageously improves the compatibility of digital micro-fluid and different application, and these application comprise (point-of-care) diagnosis from experimental analysis to point of care.

Therefore, one embodiment of the present of invention comprise the carrier (being preferably thin slice or form of film) of the reagent of prepackage and the use of digital micro-fluid device, described digital micro-fluid device comprises electrod-array, described electrod-array comprises the dispersive electrode of lining up array, described digital micro-fluid device comprises electrode controller, and described prepackage carrier comprises:

-there is the electric insulation thin slice of rear surface and hydrophobic front surface, described electric insulation thin slice is removably connected on the described electrod-array of described digital micro-fluid device, wherein said rear surface adheres to the surface of described electrod-array, described electric insulation thin slice covers described dispersive electrode, make described dispersive electrode electrically insulated from one another, and with the drop electric insulation on hydrophobic front surface

Wherein, described electric insulation thin slice has one or more reagent storage bin, and described one or more reagent storage bin are positioned on one or more pre-selected position of described hydrophobic front surface of described electric insulation thin slice;

When operation, described electrode controller can optionally start and stop described dispersive electrode drop translation is passed through to the described hydrophobic front surface of described electric insulation thin slice; And

Described one or more pre-selected position of the described hydrophobic front surface of described electric insulation thin slice is arranged to approach the drop starting on the described hydrophobic front surface of described electric insulation thin slice.

Another embodiment of the present invention provides a kind of digital micro-fluid device, comprising:

The-the first substrate, described the first substrate has and is arranged on its lip-deep electrod-array, and described electrod-array comprises the dispersive electrode of lining up array, and described digital micro-fluid device comprises the electrode controller that can optionally start and stop described dispersive electrode,

-there is the electric insulation thin slice of rear surface and hydrophobic front surface, described electric insulation thin slice is removably connected to the electrod-array (preferably described rear surface adheres to the surface of described dispersive electrode array) of described digital micro-fluid device, described electric insulation thin slice makes the dispersive electrode electrically insulated from one another of described electrod-array, and with the drop electric insulation on hydrophobic front surface, described electric insulation thin slice has one or more reagent storage bin, and described one or more reagent storage bin are positioned on one or more pre-selected position of described hydrophobic front surface of described electric insulation thin slice; Described one or more pre-selected position on described hydrophobic front surface is arranged to approach the drop starting on the hydrophobic front surface of described electric insulation thin slice;

Wherein, under the control of described electrode controller, optionally starting and stop described dispersive electrode can be by drop translation by extremely described one or more reagent storage bin of described hydrophobic front surface.

In one embodiment, apparatus of the present invention can comprise second substrate with front surface, described front surface is optionally hydrophobic surface, wherein said the second substrate and described the first substrate separate, thereby between described the first and second substrates, limit a space, described space can be included in the drop between the described front surface of described the second substrate and the described hydrophobic front surface of the above electric insulation thin slice of described electrod-array of described the first substrate.One embodiment of apparatus of the present invention can be included in the electrod-array being covered by dielectric piece on described the second substrate.In the case, the first on-chip electrod-array is optional, therefore can omit.Electric insulation thin slice also can be in the first and second substrates arbitrary or both on pre-install reagent storage bin.

The present invention also provides a kind of digital micro-fluid method, said method comprising the steps of:

-preparation has the digital micro-fluid device of electrod-array, described electrod-array comprises the dispersive electrode of lining up array, described digital micro-fluid device comprises the electrode controller being connected with the described dispersive electrode of lining up array, for applying selected voltage pattern optionally to start and to stop described dispersive electrode to described dispersive electrode, thereby liquid sample drop is passed through to described electrod-array with desirable approach on described dispersive electrode;

-removable and attachable electric insulation thin slice are provided, described electric insulation thin slice has rear surface and front working surface;

-described electric insulation thin slice is removably connected to the described electrod-array (preferably described rear surface adheres to described electrod-array) of described digital micro-fluid device, described electric insulation thin slice has hydrophobic front surface and one or more reagent storage bin, and described one or more reagent storage bin are positioned on one or more pre-selected position of described hydrophobic front surface of described electric insulation thin slice; Described one or more pre-selected position on the described hydrophobic front surface of described electric insulation thin slice is arranged to approach the drop starting on the described hydrophobic front surface of described electric insulation thin slice;

-test, guide one or more sample droplets by extremely described one or more reagent storage bin of described front working surface, by this described one or more sample droplets are delivered to described one or more reagent storage bin of being reformulated by described one or more sample droplets, and at least one the selected reagent mix comprising with described one or more reagent storage bin;

-product that any (or at least one) that form between the sample droplets of described mixing and described at least one selected reagent in described one or more reagent storage bin each (or at least one) obtained is separated; And optionally

-remove described removable and attachable electric insulation thin slice from the surface of the described electrod-array of described digital micro-fluid device, and prepare digital micro-fluid device for new test.

Can further understand function of the present invention and advantage in conjunction with the following description and drawings.Other features of the present invention and other preferred embodiments are limited by dependent claims.

Brief description of the drawings

By reference to the accompanying drawings example embodiment of the present invention is described in detail, these embodiment to scope of the present invention without any restriction.Wherein:

Figure 1A is that protein is adsorbed to digital micro-fluid device from water droplet, wherein the upper image of figure is depicted as the device before water droplet starts, is furnished with the corresponding confocal images of contre electrode, the bottom graph of figure looks like to be depicted as the same device that the drop that contains FITC-BSA (7 μ g/ml) circulates back and forth after 4 times on electrode, is furnished with the confocal images of collecting after drop moves.These two figures being done to same processing, show that confocal microscopy can be used to detect the non-specific protein absorption on apparatus surface, is the result of numeral startup.

Figure 1B is the mass spectrum of 10 μ M angiotensin Is (MW 1296).

Fig. 1 C is the cross pollution of digital micro-fluid device: the mass spectrum of 1 μ M Angiotensin II (MW 1046).Drop starts (front a kind of drop once started on this surface) on the same surface of same device, forms cross pollution with angiotensin I.

Fig. 2 is the tactful schematic diagram of the removable prepackage carrier that illustrates step by step, wherein:

(1) a slice new support with dry reagent (for plastic tab form) is fixed on digital micro-fluid device;

(2) start drop reagent by the end face of described carrier, make described drop reagent be exposed to the dry reagent of described prepackage, and make them merge, mix and cultivate, obtain chemical reaction product,

(3) after analyte non-specific adsorption, discard residue;

(4) peel the carrier with product drop or dry product off; And

(5) if wish assay products.

Fig. 3 is that the MALDI-MS that uses individual digit microfluidic device to process different analytes with different carriers analyzes:

A) 35 μ M insulin

B) 10 μ M bradykinins

C) 10 μ M 20mer DNA oligonucleotides

D) 0.01% note (ultramarker) that exceeds standard.

Fig. 4 is the analysis of prepackage carrier.There is shown the trypsase (top) and alpha-chymotrypsin (bottom) spot that apply in advance and digest the MALDI peptide mass spectrum obtaining after ubiquitin, by carry out database retrieval qualification peptide peak in MASCOT, the sequence coverage rate calculating (sequence coverage) exceedes 50%.

Fig. 5 represents to pre-install the active percentage of carrier stability analysis and the histogram of time, wherein at 1,2,3,10,20 and 30 days post-evaluation protease substrates of storage carrier (fluorine boron glimmering-casein) and interior target fluorescence, as shown in histogram, carrier is stored in-20 DEG C or-80 DEG C, and every kind of condition is calculated average response and standard deviation with 5 replicating vectors.

Fig. 6 is the different embodiment according to digital micro-fluid device of the present invention, wherein:

Fig. 6 A is the unlimited digital micro-fluid device of a side, has the carrier of a pre-installed reagents on being connected at the bottom of the first substrate;

Fig. 6 B is the unlimited digital micro-fluid device of a side, has the carrier of a pre-installed reagents and is positioned at the dielectric layer under described carrier;

Fig. 6 C is one side closed digital micro-fluid device, has the second substrate, thereby limits space or gap between the first and second substrates;

Fig. 6 D is the digital micro-fluid device of both sides sealing, has the second substrate, thereby limits space or gap between the first and second substrates.

Detailed description of the invention

Generally speaking, system described herein relates to the carrier that is more specifically applicable to the interchangeable pre-installed reagents of high throughput analysis program for digital micro-fluid device.As requested, some embodiments of the present invention disclosed herein.But, the disclosed embodiments are only to illustrate the present invention, should be appreciated that, the present invention can various interchangeable forms implement out.Accompanying drawing is not to draw in proportion, and some features may be exaggerated or dwindle to show the details of concrete part, and in order to prevent that new feature from thickening, some relevant portion may not show.So concrete structure disclosed herein and function should not be understood to have restriction, they just use essence of the present invention basis as basis and instruction those skilled in the art of claim.The carrier of the related interchangeable pre-installed reagents for digital micro-fluid device of the embodiment that illustrates, only plays instruction and unrestricted object.

Term used herein " approximately ", in the time that it uses with the size range of particle or together with other physics or chemical property or characteristic, refer to the minor variations that may exist in the upper and lower bound that comprises these size ranges, thereby do not get rid of such embodiment: on average count most of size and all meet, but statistically size likely drops on outside this scope.The object of doing is like this these embodiment not to be got rid of outside the present invention.

The basic problem that the present invention will solve is to provide a kind of device of applicable digital micro-fluid device, make digital micro-fluid device can be used in high-throughput batch processing, but avoided again the biofouling problem of the above-mentioned digital micro-fluid device of discussing in background of invention part simultaneously.How debatable discuss biofouling is, the inventor has carried out various research and determine the scope of this problem.

the protein adsorption of digital micro-fluid device and cross pollution analysis

Evaluate lip-deep protein adsorption with confocal microscopy.Generally speaking, a drop that contains 7 μ g/mlFITC-BSA is moved to digital micro-fluid device.Before and after starting this drop, spot is taken a picture, obtain two images.Between the drop starting period, due to non-specific protein absorption, on surface, leave residue, can be detected by confocal microscopy.These residues can cause digital micro-fluid device to produce two class problems:

(1) surface may become toughness, hinders drop to move, and

(2), if carry out multiple experiments, may produce cross-contamination issue.

The present invention uses is furnished with Ar ⁺(488nm) the FluoView 300 scanning confocal microscope (OLYMPUS of laser instrument, Markam, ON), be adsorbed on the protein (Figure 1A) of digital micro-fluid apparatus surface in conjunction with 100 times of object lens (numerical aperture is 0.95) analysis.By 510-525nm bandpass filter, use FluoView image acquisition software (OLYMPUS) to form each digital picture from the average of four picture frames the fluorescence of the protein of absorption mark.

Evaluate the amount of cross-contamination of two different peptide samples that are activated the identical approach by same device with MALDI-MS.Particularly, start for the first time 2 μ l 10 μ M angiotensin I drops, start for the second time 2 μ l1 μ M Angiotensin II drops.The collection of illustrative plates of the angiotensin I producing after startup for the first time as shown in Figure 1B, is quite clean; But the collection of illustrative plates that as shown in Figure 1 C, Angiotensin II produces is subject to the last residue starting to be polluted.These test in, digital micro-fluid start after, sample droplets is transferred into MALDI target and carries out crystallization and analysis, means that cross pollution comprises: (a) for the first time start adsorption step, and (b) for the second time start desorption.The intensity of estimating angiotensin I pollutant is approximately 10% of Angiotensin II highest peak (MW 1046).This approximately corresponding to 1% or 0.1 μ M angiotensin I at the non-specific dirt of digital micro-fluid device.Even if the viscosity ratio protein of test peptides is little, the numerical value (being less than 8%FITC-BSA is adsorbed on digital micro-fluid device) consistent (article " Pluronic additives:A solution to sticky problems in digital the microfluidics, " Langmuir24:6382-6389 delivering for 2008 referring to people such as Luk) that this result is also reported with Luk.Except polluting, because having hindered drop, the non-specific adsorption of last time startup moves glibly, especially in the start-up course of Angiotensin II sample.Therefore, need higher starting resistor to impel drop to move through next group electrode.But owing to being bonded at firmly, to become the motionless situation of for good and all adhering on contaminated surface also unworkable for drop for this method, increase starting resistor this situation is had no to help, if let alone, voltage is too high may cause puncturing dielectric and destroying device.

the disposable carrier of interchangeable prepackage

The invention provides the disposable carrier of interchangeable prepackage, on these carriers, reagent has on the pre-selected position that is positioned at upper surface tactfully.These carriers can be used as replaceable carrier and use together with digital micro-fluid device, and these carriers are added on the electrod-array of digital micro-fluid device.

Referring to Fig. 2, there is shown the disposable electric insulation carrier 10 according to prepackage of the present invention, this carrier 10 has a prepackage reagent storage bin 12 on the hydrophobic front surface that is arranged on electric insulation thin slice 11.Described disposable carrier 10 can be any thin dielectric piece or film, as long as it keeps chemically stable to the reagent of prepackage.For example, can use any polymer plastic as saran wrap (saran wrap).Except plastic food packing film, comprise generally/office's adhesive tape of other carriers and tighten paraffin sheet and also can be used as interchangeable digital micro-fluid carrier.

Disposable carrier 10 is connected to the electrod-array 16 of digital micro-fluid device 14, wherein the rear surface of carrier 10 invests electrod-array 16, the reagent storage bin 12 being placed on the surface (reagent droplet can translation by this surface) of carrier 10 is aimed at previously selected indivedual electrodes 18 of electrod-array 16, as shown in the step of Fig. 2 (1) and (2).Before test, two reagent droplet 20 and 22 are dropped on device.The most handy suction pipette head 36 being connected with sample container 32 or solvent container 34 drops in drop 20 and 22 on device and (sees Fig. 2).Another kind of selection is that container 32 can be connected with device with 34 or be one-body molded with device, makes to start digital micro-fluid and just can distribute drop 20 and 22.

As shown in the step of Fig. 2 (3), reagent droplet 20 and 22 is activated by the end face of disposable thin slice or carrier in test, helps reagent droplet 20 and 22 on electrode, mix and merge with desirable reagent storage bin 12.After reaction completes, can as shown in step (4), peel off disposable carrier 10, if wish, can as shown in step (5), analyze the product 26 obtaining.Next round is connected to new disposable carrier 10 on digital micro-fluid device 14 while analysis again.When removable carrier is still connected on digital micro-fluid device 14, also can analyze product 26.Use other prepackage carrier can repeat said process.In addition, if the drop that contains product can be by division, contain cell with other droplets mixings and/or they and can cultivate together with cell culture medium.

Because the residue of testing on the disposable thin slice of front a slice or carrier 10 28,30 can be along with disposable carrier 10 is removed, described in avoided cross pollution.Above-mentioned test is undertaken by a kind of prepackage reagent storage bin 12, but it will be understood by those skilled in the art that the carrier 10 of prepackage can load onto plurality of reagents, carries out continuously or parallel test with multiple drop reagent 20 and 22.

In an embodiment of the present invention, electric insulation thin slice 11 and the electrod-array 16 of prepackage can comprise the alignment mark that in the time that electric insulation thin slice 11 is connected to electrod-array 16, electric insulation thin slice 11 and electrod-array 16 is aimed at separately, so that the upper one or more pre-selected of the front working surface 11a of electric insulation thin slice 11 position 13 is chosen and discrete starting electrode 18 registrations of one or more pre-selected of electrod-array.When reagent storage bin 12 and electrode 18 registrations of pre-selected, they can be positioned at selected electrode above or in the horizontal close to selected electrode, make it on the gap between adjacent electrode.

Fig. 6 A is depicted as the unlimited digital micro-fluid device of a side with carrier 10, and wherein carrier 10 has been pre-installed reagent storage bin 12 and used together with digital micro-fluid device 14, and carrier 10 is connected to the first substrate 24.Digital micro-fluid device comprises the array 16 and the electrode controller 19 that are made up of dispersive electrode 17.Prepackage carrier 10 comprises the electric insulation thin slice 11 with hydrophobic front surface 11a and rear surface 11b.Electric insulation thin slice 11 is removably connected on the surface 16 ' of electrod-array 16 of digital micro-fluid device 14.In the time that electric insulation thin slice 11 is placed on the electrod-array 16 of digital micro-fluid device 14, it covers described dispersive electrode 17 and makes the mutual electric insulation of described dispersive electrode 17, also with hydrophobic front surface 11a on drop 20,22,33 electric insulations.The electric insulation thin slice 11 of first embodiment of the invention has one or more reagent storage bin 12, and described one or more reagent storage bin 12 are positioned on one or more pre-selected position 13 of hydrophobic front surface 11a of electric insulation thin slice 11.When operation, the electrode controller 19 of digital micro-fluid device 14 can optionally start and stop described dispersive electrode 17 by drop 20,22,33 translations are by the hydrophobic front surface 11a of electric insulation thin slice 11, and the upper one or more pre-selected of the front working surface 11a of electric insulation thin slice 11 position 13 is configured to approach the upper drop 20 starting of hydrophobic front surface 11a of electric insulation thin slice 11,22,33.

Preferably, the surface 16 ' that described electric insulation thin slice 11 can connect or be connected to electrod-array 16 by adhesive 15 is upper, and wherein adhesive 15 contacts the surface 24 ' of the rear surface 11b of electric insulation thin slice 11 and the surface 16 ' of electrod-array 16 and/or the first substrate 24.More preferably, described electric insulation thin slice 11 is included in the adhesive 15 on the 11b of rear surface described in it, and described adhesive 15 can contact described electrod-array, and described electric insulation thin slice is adhered to described the first substrate 24.

Fig. 6 B is depicted as the unlimited digital micro-fluid device of a side, and described digital micro-fluid device has one and pre-installed the carrier of reagent and the dielectric layer under this carrier.Digital micro-fluid device 14 (described similar with Fig. 6 A) comprises that key character is as electrode controller 19; In addition, also comprise the drop 20,22,33 for the treatment of translation.But in the embodiment shown in Fig. 6 B, adhesive 15 only contacts the surface 24 ' of rear surface 11b and first substrate 24 of electric insulation thin slice 11; Or adhesive 15 can be arranged on the upper (not shown) of whole rear surface 11b of electric insulation thin slice 11.In the present embodiment, digital micro-fluid device 14 preferably comprise be directly added in described electrod-array 16 described surperficial 16 ' on dielectric layer 25, it is clipped between described electrod-array 16 and described electric insulation thin slice 11.

Fig. 6 C is depicted as one side closed digital micro-fluid device, and described digital micro-fluid device has the second substrate, and described the second substrate limits space or the gap between the first and second substrates.Digital micro-fluid device 14 (described similar with Fig. 6 B) comprises that key character is as electrode controller 19; In addition, also comprise the drop 20,22,33 for the treatment of translation.In the present embodiment, digital micro-fluid device 14 preferably also comprises the second substrate 27, and it is optionally the front surface 27 ' of hydrophobic surface that described the second substrate 27 has.The second substrate 27 and the first substrate 24 separate, thereby define a space or gap 29 between the first substrate 24 and the second substrate 27, described space or gap 29 can be included in the drop 20 that the front surface 27 ' of the second substrate 27 and the described electrode 16 of described the first substrate 24 power between the hydrophobic front surface 11a of heat insulating lamella 11,22,33.Preferably, electrode controller 19 is also controlled the electrostatic charge on the surface 27 ' of the second substrate.Contrary with Fig. 6 B, the adhesive 15 of the present embodiment only contacts the rear surface 11b of electric insulation thin slice 11 and is positioned at the dielectric layer 25 on the surface 16 ' of electrod-array 16 of the first substrate 24.Or adhesive 15 can be arranged on the whole rear surface 11b of electric insulation thin slice 11.

Fig. 6 D is depicted as the digital micro-fluid device of both sides sealing, and described digital micro-fluid device has the second substrate, and described the second substrate limits space or the gap between the first and second substrates.Digital micro-fluid device 14 (described similar with Fig. 6 A-6C) comprises the array 16 and the electrode controller 19 that are made up of dispersive electrode 17.Prepackage carrier 10 comprises the electric insulation thin slice 11 with hydrophobic front surface 11a and rear surface 11b.Electric insulation thin slice 11 is removably connected on the surface 16 ' of the first electrod-array 16 of digital micro-fluid device 14.In the present embodiment, digital micro-fluid device 14 preferably also comprises second substrate 27 with front surface 27 '.According to the present embodiment, the front surface 27 ' of the second substrate 27 is not hydrophobic, and it comprises the second extra electric insulation thin slice 31 with rear surface 31b and hydrophobic front surface 31a.Described the second extra electric insulation thin slice 31 is removably connected on the front surface 27 ' of the second substrate 27, and wherein rear surface 31b is adhered to front surface 27 '.Not or have one or more reagent storage bin 12, described one or more reagent storage bin 12 are not positioned on one or more pre-selected position of hydrophobic front surface 31a of the second extra electric insulation thin slice 31 described the second extra electric insulation thin slice 31.

Contrary with Fig. 6 B, the adhesive 15 of the present embodiment only contacts the rear surface 11b of electric insulation thin slice 11 and is positioned at the surface 16 ' of the electrod-array 16 of the first substrate 24.In a relative side, adhesive 15 is arranged on the whole rear surface 31b of the second extra electric insulation thin slice 31.Or adhesive 15 can be arranged on the upper (not shown) of whole rear surface 11b of electric insulation thin slice 11.Preferably (as shown in Figure 6 D), digital micro-fluid device 14 comprises the upper extra electrod-array 35 of front surface 27 ' that is arranged on the second substrate 27, and the second extra electric insulation thin slice 31 that described extra electrod-array 35 is had hydrophobic front surface 31a covers.As shown in Fig. 6 B and 6C, the digital micro-fluid device 14 of Fig. 6 D preferably also comprise be directly added in described electrod-array 35 described surperficial 27 ' on dielectric layer 25, it is clipped between described electrod-array 35 and described the second electric insulation thin slice 31.Between electric insulation thin slice 11 and the surface 16 ' of electrod-array 16, also can place another dielectric layer 25 (not shown).(not shown) in an alternative embodiment, the described extra electrod-array 35 on the second substrate 27 applies hydrophobic coating, and the second electric insulation layer 31 is not set.

Disposable carrier 10 can be packed and sell together with reagent storage bin together with multiple other carriers, and wherein said reagent storage bin contains one or more selected reagent of being used as concrete test type.Carrier 10 in packaging can be with the prepackage reagent storage bin 12 of similar number, and each reservoir comprises identical reagent composition.Reagent storage bin preferably comprises dry reagent, but also can comprise sticking gel reagents.

Potential application of the present invention is to cultivate and analysis of cells in reagent storage bin.In such application, reagent storage bin can comprise the attachment element of biological substrate and adherent cell, as fibronectin, collagen, laminin, polylysin etc. and combination thereof.The drop that contains cell can be directed to biological substrate reservoir, for adherent cell permissive cell adhere to.After adhering to, can in digital micro-fluid device, cultivate or analysis of cells.

Although the electrod-array 16 that the digital micro-fluid device 14 shown in Fig. 2 has single substrate 24 and forms thereon, those skilled in the art should be understood that, digital micro-fluid device can comprise the second substrate 27, it is optionally the front surface 27 ' of hydrophobic surface that this second substrate 27 has, wherein the second substrate and the first substrate are isolated, thereby between the first and second substrates, define a space, described space can be included in the drop (seeing Fig. 6 C) that the front surface of the second substrate and the described electrod-array of the first substrate power between the hydrophobic front surface of heat insulating lamella.The second substrate can be substantial transparent.Except the embodiment that Fig. 6 C describes, prepackage carrier 10 (comprising the first electric insulation thin slice 11 with hydrophobic front surface 11a and rear surface 11b) can removably be connected on the surface 27 ' of the second substrate 27 of digital micro-fluid device 14.Meanwhile, electrod-array 16 can apply not removable electrical insulator (not shown).

When the front surface of the second substrate is not hydrophobic, device can comprise the extra electric insulation thin slice with rear surface and hydrophobic front surface, described extra electric insulation thin slice is removably connected on the front surface of the second substrate, and wherein its rear surface is adhered to the front surface of the second substrate.Described extra electric insulation thin slice has one or more reagent storage bin, and described one or more reagent storage bin are positioned on one or more pre-selected position of hydrophobic front surface of described electric insulation thin slice.

In addition, on the front surface 27 ' of the second substrate 27, extra electrod-array 35 can be installed, and comprise and be added in the one deck on extra electrod-array 35 with hydrophobic front surface, described one deck being added on extra electrod-array 35 has hydrophobic front surface 31a, it can be extra electric insulation thin slice 31, extra electric insulation thin slice 31 has one or more reagent storage bin 12, and described one or more reagent storage bin 12 are positioned on one or more pre-selected position 13 of described hydrophobic front surface.In two plate designs shown in Fig. 6 D, the first substrate 24 optionally can be without prepackage heat insulating lamella or carrier 11 that reagent storage bin 12 has been installed.

The present invention and the present invention describe in connection with following research and embodiment for the effect of high throughput test, but they only work the effect of illustrating, and are nonrestrictive.

Detailed experiment

reagent and material

The working solution of the whole matrix of preparation (α-CHCA, DHB, HPA and SA) in 50% AG acetonitrile/deionized water (v/v) and 0.1%TFA (v/v), concentration is 10mg/ml, and these working solutions are stored in 4 DEG C under dark condition.It is standby that the stock solution (10 μ M) of angiotensin I, II and bradykinin spends ion water making, and work buffer solution (10mM Tris-HCl for the stock solution of ubiquitin and myoglobins (100 μ M), 1mM CaCl20.0005%w/v blocked polyethers F68, pH 8) preparation.All standard inventory solution all stores at 4 DEG C.Work buffer solution preparation for the stock solution (100 μ M) of digestive ferment (bovine trypsin and alpha-chymotrypsin), and store until use with aliquot at-80 DEG C.Before test, just standard liquid and enzyme are heated to room temperature and use deionized water (peptide) and working buffer liquid (protein, enzyme and fluorometric reagent) dilution.Before using, just prepare fluorescent test solution (3.3 μ M cancellation fluorine boron in working buffer liquid glimmering-casein and 2 μ M rhodamine Bs).

making and the operation of device

Adopt standard micro manufacturing technology to make digital micro-fluid device, in this device, 200nm hard chromium electrode forms pattern on glass substrate.Before experiment, device is loaded onto (a) unmodified carrier, or (b) the pre-carrier of installed reagents.In the time using unmodified carrier (a), drip several silicone oil to electrod-array, then cover vinyl cover.Then spin coated Teflon-AF (1%w/w fluoridize in liquid Fluorinert FC-40,1000RPM, 60s) from the teeth outwards, and with heating plate annealing (75 DEG C, 30min).In the time using prepackage carrier (b), before being added in device, first vinyl cover is modified.Modification comprises three steps: vinyl cover sticked on the glass substrate that does not form pattern, and coating Teflon-AF (the same), and add reagent storage bin.A rear step can realize like this: 2 μ l enzymes (6.5 μ M trypsase or 10 μ M alpha-chymotrypsins) drop is dripped from the teeth outwards, and allows droplet drying with pipette.Prepackage carrier can use immediately, or is sealed in sterilized plastic culture dish and is stored in-20 DEG C.Before use, prepackage carrier is heated to room temperature (words if necessary), the substrate that never forms pattern peels, be then added on the electrod-array of coating silicone oil, then with heating plate annealing (75 DEG C, 2min).Except food package film, plastic adhesive tape and paraffin also can be used to be arranged on device.With finger pressure, adhesive tape is connected on device lightly, and it is thick that paraffin is stretched to about 10mm, then coiler device, forms bubble-free sealing.

1mm × 1mm electrode of device is made the design of " Y " shape, and wherein the gap between each electrode is 10 μ m.To continuous electrode to applying driving voltage (400-500V _rMS), make 2 μ l drops move and merge on the device with opening panel pattern (without top cover) operation.The output of the function generator that is 18kHz by operating frequency is amplified, and produces driving voltage, more described driving voltage is added on the contact exposing with staff.Monitor and record the startup of drop by CCD camera.

analyze with MALDI-MS

Use matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to evaluate the sample starting on digital micro-fluid device.Matrix/sample spot is made two kinds of patterns: routine and original position.In normal mode, sample device on operation, with pipette collect and sample is dispensed on stainless steel target.Add matrix solution, allow merge after droplet drying.In pattern, make the dispersant liquid drop that contains sample and matrix move, merge and initiatively mix by digital micro-fluid, then in dry tack free in position.Relating in the original position experiment of pre-installing carrier, first carry out chip reaction and implement again matrix/crystallization: the drop that contains sample protein is driven and moves to the dry spot point that contains digestive ferment (trypsase and alpha-chymotrypsin).Cultivating together with enzyme after (room temperature, 15min), a matrix is urged to spot, cancellation reaction, allows the droplet drying merging.After cocrystallization, by carrier stripping off device carefully, be then fixed on stainless steel target with two-sided tape.Different analytes uses different matrix: peptide standard and digestive juice adopt α-CHCA, and the note that exceeds standard adopts DHB, and oligonucleotides adopts HPA, and protein adopts SA.At least three of each sample evaluations copy spot.

With MALDI-TOF Micro-MX MS mass spectrograph (Waters, Milford, the MA) analyzing samples of forward mode (positive mode) operation.The evaluation of peptide standard and digestive juice is carried out in reflective-mode (reflectron mode), and mass-to-charge ratio (m/z) scope is 500-2, and 000.The evaluation of protein is carried out in linear model (linear mode), and mass-to-charge ratio (m/z) scope is 5,000-30, and 000.Each collection of illustrative plates is collected at least 100 spots, and adjustment laser power, reaches to the best signal to noise ratio (S/N).Then, be normalized, go baseline to carry out deal with data to analyte maximum peak, and do smoothing by 15 moving averages (15-point running average).Pack retrieval SwissProt database and come the collection of illustrative plates of enzyme analysis digestive juice by Mascot identification of proteins.Database retrieval obtains the leakage of 1 permission and cuts site (allowed missed cleavage), and mass spectrum accuracy is +/-1.2Da, there is no other modifications.

peptide/protein on replaceable carrier is made to MS to be analyzed

In order to illustrate this new strategy, process four kinds of dissimilar analytes with individual digit microfluidic device, new removable carrier is all used in each processing.As shown in Figure 3, four kinds of analytes comprise insulin (MW 5733), bradykinin (MW 1060), 20-mer oligonucleotides (MW 6135) and synthetic polymer note 1621 (MW900-2200) that exceed standard.Each removable carrier is analyzed by MALDI-MS original position, does not observe the evidence of any cross pollution.In applicant's laboratory, conventional equipment normally disposable (use and once abandon); But in the experiment of the removable carrier of use, the device of use can carry out 9-10 test conventionally, but performance does not decline.Therefore,, except not having the problem of cross pollution, movable carrier strategy also can reduce widely supports the needed making of digital micro-fluid and assembling burden.

Except plastic food packing film, other carriers comprise office's adhesive tape and tighten cere sheet and also can be used as interchangeable carrier.The same with food package film, find that the carrier being formed by adhesive tape and cere sheet can support drop to move and be conducive to device Recycling (data do not show).In addition, the carrier being formed by these materials is favourable, reason be they use before without annealing in process.But some other aspect declines the attraction of these materials.Covering of being formed by adhesive tape easily destroys starting electrode (although be to infer, low adhesive tape may not have this problem) after reusing.In addition, because the tape carrier of testing is relatively thick, (～45 μ m), need larger driving electricity (～900V _rMS) operate drop.On the contrary, the thickness of tightening cere sheet is about 10 μ m, and driving voltage is with few by the film formed carrier of packaging for foodstuff voltage difference used.But, the carrier thickness that this mode forms is found to be inhomogeneous, and the reliability that moves them for drop is poor.In brief, various carrier all conforms to the concept of removable lid, but because behaving oneself best at present for the carrier that formed by food package film, so test with this material.

Two shortcomings of removable carrier strategy are that voids and material are incompatible.In initial experiment, in use procedure, often can between carrier and apparatus surface, see the bubble being trapped.In the time that driving voltage is added near near electrode voids, observe electric arc, this can damage device.Find, using before plastic sheeting with the several silicone oil apparatus surface of getting wet, can address this problem.After annealing, silicone oil evaporates, and is left to seal without bubble.To be that material is incompatible more attract people's attention a rear problem.If use aggressiveness solvent, the material in carrier may be lost in solution, interference test.In experiment of the present invention, in MALDI-MS spectrum, do not find pollution peak (comprising the contrast mass spectrum being produced by naked carrier surface, not shown), but cannot get rid of the possibility that middle this problem of existence is set at other.Since various materials can both be used for forming carrier (on seeing), the applicant is confident of can using other substitutes in the inapplicable situation of food package film of coating Teflon.

prepackage carrier and stability analysis thereof

To overcome in the process of dirt and cross pollution, have recognized that this technology can also become the basis of an infusive innovation of digital micro-fluid at the replaceable carrier strategy of research.Reagent is deposited on to (and several such carriers are provided) on carrier in advance, this strategy is by one of the quick introducing device of reagent new platform easily by digital micro-fluid technical transform, also can solve the known external world of microfluid to the problem of chip interface (seeing article " Solving the " World-to-chip " Interface problem with a microfluidic matrix " the Analytical Chemistry 75:4718-4723 that the people such as article " A high-throughput continuous sample introduction interface for microfluidic chip-based capillary electrophoresis systems " Analytical Chemistry 74:1223-1231 and Liu that the people such as Fang delivered in 2002 delivered in 2003).

In order to illustrate this New Policy, prepare the food package film of doing to have in advance dry digestive ferment spot, then with digital micro-fluid, the drop that contains simulation substrate ubiquitin is delivered to these spots.Cultivating after one section of appropriate time, the drop that contains MALDI matrix is delivered to these spots, then spot is dried and annealing.As shown in Figure 4, MALDI mass spectrum is consistent with the peptide quality fingerprinting expected result of analyte.In fact, in the time evaluating with proteomics search engine MASCOT, performance is excellent, the sequence coverage rate of all tests be 50% or more than.

In the prepackage carrier strategic process of optimization protease test, it is quite reliable having observed this method.First,, using blocked polyethers F68 as solution additive, help analyte drop (being ubiquitin) mobile herein; This reagent has been found to reduce the ionizing efficiency (referring to people such as Boemsen at 1997 articles of delivering " Infuence of solvents and detergents on matrix-assisted laser desorption/ionization mass spectrometry measurements of proteins and oligonucleotides " Rapid Communications in Mass Spectrometry 11:603-609) of MALDI-MS.Fortunately, consumption (0.0005%w/v) is herein very low, so that do not observe this effect.Secondly, seldom see trypsase and alpha-chymotrypsin from dissolved peak, the applicant is by low it ratio owing to enzyme-to-substrate and the reaction time is short.The 3rd, in preliminary test, determine that annealing steps (75 DEG C, 2min) can not affect the activity of dry enzyme.If use the reagent to these condition responsives later, the applicant's program evaluation for example, by the carrier that does not need the material (low adhesive tape) of annealing to make.In any case, the prompting of the unfailing performance of these preliminary tests, this strategy finally can be applied in field widely, as immunoassay or microarray analysis.

As mentioned above, prepackage carrier strategy (is shown in the people such as Linder, 2005 with the concept that stores pre-installed reagents in microchannel is similar; The people such as Hatakeyama, 2006; The people such as Zheng, 2005; The people such as Furuberg, 2007; The people such as Garcia, 2004; The people such as Zimmermann, 2008; And the people such as Chen, 2006 " Microfluidic cartridges preloaded with nanoliter plugs of reagents:An alternative to 96-well plates for screening " Current Opinion in Chemical Biology 10:226-231).Conventionally after use, to throw aside device different from existing method, and in prepackage carrier strategy of the present invention, basic device framework can be recycled and reused in a large amount of tests.In addition, because reagent (with the product obtaining) is not all encapsulated in passage, so their form is to be convenient to very much analyze in essence.For example, in this research, this form facilitates MALDI-MS to detect, but estimates can use large-scale all kinds of detector, for example optical reader and acoustic sensor in the future.Finally, although this Proof-Of Principle (proof-of-principle) research and utilization is with the packaging for foodstuff membrane carrier of single reagent spot, estimation available microarray speckled ware in future (microarray spotter) is made with the preloaded of many different reagent and is carried out multinomial analysis.

Be used for practical application, prepackage carrier must keep active between the storage life.Evaluate the storage life of these reagent spots, the applicant has implemented a quantitative proteopepsis test.Reporter in this test is the casein of the glimmering mark of cancellation fluorine boron, has low fluorescence when complete, but after digested, has high fluorescence.In this prepackage reagent stability test, the drop that contains reporter is driven to prepackage trypsase spot, after cultivation, (described above with the fluorescence signal in plate reader measurement drop, see article " Pluronic additives:A solution to sticky problems in digital microfluidics, " Langmuir 24:6382-6389 that the people such as Luk delivered in 2008; Article " Digital microfluidics for cell-based assays " the Lab on a Chip 8:519-526 that the people such as Barbulovic-Nad delivered in 2008; Article " A digital microfluidic approach to homogeneous enzyme assays " the Analytical Chemistry80:1614-1619 that Miller and Wheeler delivered in 2008).Using in the preliminary experiment of the new prepackage carrier of making, determine, the in the situation that of concentration used, reaction completed in 30 minutes.With interior mark rhodamine B correction correction error, evaporative effect and the zero creep along with passage of time generation.

In storage life experiment, prepackage carrier stores different time (1,2,3,10,20 or 30 day) at-20 DEG C or-80 DEG C.In each experiment, after the carrier that thaws, it is placed on device, drop is urged to trypsase and cultivates 30 minutes, record report/interior mark signal ratio.At least 5 different carriers of every kind of average evaluation.As shown in Figure 5, storage life performance is that excellent-carrier stores the long original activity that still can preserve > 75% for 30 days at-80 DEG C.Original activity that carrier still can be preserved > 50%-20 DEG C of same times of storage.This species diversity may be due to average storage temperature difference, or has reflected that the use state of-20 DEG C of freezers is from defrost mode (often having temperature fluctuation), and the temperature of-80 DEG C of freezers is invariable.In any case, in preliminary test, the performance of these carriers is excellent, can predict, pH or the ionic strength of regulatory enzyme buffer suspension liquid, or add stabilizing agent as trehalose, a kind of disaccharides (seeing article " Stability of monoclonal igm antibodies freeze-dried in the presence of trehalose " the Journal of Immunological Methods 181:37-43 that the people such as Draber delivered in nineteen ninety-five) being widely used at industrial preservation dry state protein, in the limit that can extend shelf life in the future.

Briefly, the inventor has worked out a new digital micro-fluid strategy, in fact contributes to ad infinitum to reuse various devices, and without the problem of worrying that fork pollutes, and can replace rapidly pre-installed reagents.The present invention can be converted into digital micro-fluid the versatile platform of chip lab (lab-on-a-chip) application.

The term herein adopting " comprises " and " comprising " should be interpreted as open comprising and comprise, non-removing property.Particularly, use term " to comprise " and when " comprising " and modification thereof, refer to and comprise specific feature, step or composition when description comprises claims.These terms should not be understood to get rid of the existence of other features, step or composition.

Above-mentioned description of the preferred embodiment of the present invention just illustrates principle of the present invention, is not the present invention is limited to these preferred embodiments.All embodiment that protection scope of the present invention is contained by appended claims and equivalent feature thereof limit.

Identical feature represents with identical Reference numeral, even if these Reference numerals only occur in the drawings and do not particularly point out in description.

Reference numeral:

10 disposable prepackage carriers

11 electric insulation thin slices

The hydrophobic front surface of 11a electric insulation thin slice; Front working surface

The rear surface of 11b electric insulation thin slice

12 prepackage reagent storage bin

13 pre-selected positions

14 digital micro-fluid devices

15 adhesives

16,16 ' electrod-array; The surface of electrod-array

17 dispersive electrodes

Indivedual electrodes of 18 pre-selected

19 electrode controllers

20 reagent droplet

21 alignment marks

22 reagent droplet

23 form the conductive coating of pattern

The 24,24 ' first substrate; The surface of the first substrate

25 dielectric layers

26 product that obtain

The 27,27 ' second substrate; The surface of the second substrate

28 previous test residues

29 spaces

30 previous test residues

31 extra electric insulation thin slices

31a, the hydrophobic front surface of the extra electric insulation thin slice of 31b; The rear surface of extra electric insulation thin slice

32 sample container

33 solvent droplets

34 solvent containers

The 35,35 ' extra electrod-array; The surface of extra electrod-array

36 suction pipette heads

Claims

1. the carrier (10) of pre-installed reagents using with digital micro-fluid device (14), described prepackage carrier (10) has the one or more reagent storage bin (12) that are positioned on one or more pre-selected position (13), and described prepackage carrier comprises the electric insulation thin slice (11) with hydrophobic front surface (11a) and rear surface (11b); Described digital micro-fluid device (14) comprises by dispersive electrode (17) arranges the electrod-array (16) and the electrode controller (19) that form, described electrode controller (19) can optionally start and stop described dispersive electrode (17) drop is moved on described hydrophobic front surface to the described one or more pre-selected position (13) on the hydrophobic front surface (11a) of described electric insulation thin slice (11)

It is characterized in that described electric insulation thin slice (11):

(a) before described reagent storage bin (12) is arranged on the hydrophobic front surface (11a) of described electric insulation thin slice (11) being connected to digital micro-fluid device (14);

(b) rear surface (11b) of described electric insulation thin slice (11) can be connected on the surface (16 ') of described electrod-array (16) of digital micro-fluid device (14);

(c) in the time being placed on described electrod-array (16), described electric insulation thin slice (11) covers described dispersive electrode (17), make described dispersive electrode (17) electrically insulated from one another, and with the drop (20 on described hydrophobic front surface (11a), 22,33) electric insulation;

(d) can peel from the described surface (16 ') of described electrod-array (16) so that carry out optional analysis and abandon; And

(e), in the time that the described electrod-array (16) from described digital micro-fluid device (14) is peeled off, by connecting upper new prepackage carrier (10), described electrod-array (16) can be reused.

2. carrier as claimed in claim 1 (10), it is characterized in that: the described surface (16 ') that described electric insulation thin slice (11) can connect or be connected to described electrod-array (16) by adhesive (15) is upper, and wherein said adhesive (15) contacts the surface (24 ') of the described rear surface (11b) of described electric insulation thin slice (11) and the described surface (16 ') of described electrod-array (16) and/or the first substrate (24).

3. carrier as claimed in claim 1 or 2 (10), it is characterized in that: described electric insulation thin slice (11) and described electrod-array (16) or the first substrate (24) comprise the alignment mark (21) that makes described electric insulation thin slice (11) and described electrod-array (16) aim in the time that described electric insulation thin slice (11) is connected to described electrod-array (16) separately, so that the above one or more pre-selected position (13) of the described hydrophobic front surface (11a) of described electric insulation thin slice (11) is chosen and indivedual electrodes (18) of one or more pre-selected of described electrod-array (16) are overlapping.

4. carrier as claimed in claim 1 or 2 (10), is characterized in that: described electric insulation thin slice (11) comprises the material that is selected from polymer, plastics and wax.

5. carrier as claimed in claim 1 or 2 (10), it is characterized in that: described electric insulation thin slice (11) is with the conductive coating (23) that forms pattern, and described conductive coating (23) can provide reference or starting resistor to described electrod-array (16).

6. carrier as claimed in claim 1 or 2 (10), it is characterized in that: described one or more reagent storage bin (12) comprise single kind of reagent or at least two kinds of reagent, in every kind of situation, reagent is selected from the group that comprises dry reagent or viscogel reagent.

7. carrier as claimed in claim 6 (10), it is characterized in that: described one or more reagent storage bin (12) are to exceed a reagent storage bin, and wherein in each reagent storage bin (12), at least one reagent is different from the reagent that at least one reservoir in other all reagent storage bin comprises.

8. the carrier (10) as described in any one in claim 1,2 or 7, it is characterized in that: described electric insulation thin slice (11) is included in the adhesive (15) on described rear surface (11b), described adhesive can contact described electrod-array makes described electric insulation thin slice (11) adhere to the first substrate (24).

9. a digital micro-fluid device (14), comprising:

(a) the first substrate (24), described the first substrate (24) has to be arranged on its surface (24 ') arranges by dispersive electrode (17) electrod-array (16) forming;

(b) electrode controller (19), described electrode controller (19) can optionally start and stop the described dispersive electrode (17) of described electrod-array (16); And

(c) carrier according to claim 1 (10), described carrier (10) is included in the electric insulation thin slice (11) of hydrophobic front surface (11a) the one or more reagent storage bin of upper prepackage (12).

10. digital micro-fluid device as claimed in claim 9 (14), it is characterized in that: the surface (16 ') that described electric insulation thin slice (11) can connect or be connected to described electrod-array (16) by adhesive (15) is upper, and wherein said adhesive (15) contacts the surface (24 ') of the described rear surface (11b) of described electric insulation thin slice (11) and the surface (16 ') of described electrod-array (16) and/or the first substrate (24).

11. digital micro-fluid devices (14) as described in claim 9 or 10, it is characterized in that: described electric insulation thin slice (11) and described electrod-array (16) or the first substrate (24) comprise the alignment mark (21) that makes described electric insulation thin slice (11) and described electrod-array (16) aim in the time that described electric insulation thin slice (11) is connected to described electrod-array (16) separately, so that the above one or more pre-selected position (13) of the described hydrophobic front surface (11a) of described electric insulation thin slice (11) is chosen and indivedual electrodes (18) of one or more pre-selected of described electrod-array (16) are overlapping.

12. digital micro-fluid devices (14) as described in claim 9 or 10, is characterized in that: described electric insulation thin slice (11) comprises the material that is selected from polymer, plastics and wax.

13. digital micro-fluid devices (14) as described in claim 9 or 10, it is characterized in that: described electric insulation thin slice (11) is with the conductive coating (23) that forms pattern, and described conductive coating (23) can provide reference or starting resistor to described electrod-array (16).

14. digital micro-fluid devices (14) as described in claim 9 or 10, it is characterized in that: described one or more reagent storage bin (12) comprise single kind of reagent or at least two kinds of reagent, in every kind of situation, reagent is selected from the group that comprises dry reagent or viscogel reagent.

15. digital micro-fluid devices as claimed in claim 14 (14), it is characterized in that: described one or more reagent storage bin (12) are to exceed a reagent storage bin, and wherein in each reagent storage bin (12), at least one reagent is different from the reagent that at least one reservoir in other all reagent storage bin comprises.

16. digital micro-fluid devices (14) as described in any one in claim 9,10 or 15, it is characterized in that: described electric insulation thin slice (11) is included in the adhesive (15) on described rear surface (11b), described adhesive can contact described electrod-array makes described electric insulation thin slice (11) adhere to described the first substrate (24).

17. digital micro-fluid devices (14) as described in any one in claim 9,10 or 15, it is characterized in that: described device comprises the dielectric layer (25) on the described surface (16 ') that is directly added in described electrod-array (16), it is clipped between described electrod-array (16) and described electric insulation thin slice (11).

18. as claim 9, digital micro-fluid device (14) in 10 or 15 described in any one, it is characterized in that: described device also comprises the second substrate (27), it is optionally the front surface (27 ') of hydrophobic surface that described the second substrate (27) has, wherein said the second substrate (27) separates with described the first substrate (24), thereby define a space (29) between described the first substrate (24) and described the second substrate (27), described space (29) can be included in the drop (20 between the described front surface (27 ') of described the second substrate (27) and the described hydrophobic front surface (11a) of the above electric insulation thin slice (11) of described electrod-array (16) of described the first substrate (24), 22, 33).

19. digital micro-fluid devices as claimed in claim 18 (14), is characterized in that: described the second substrate (27) is transparent substantially.

20. digital micro-fluid devices as claimed in claim 18 (14), it is characterized in that: the described front surface (27 ') of described the second substrate (27) is not hydrophobic, it comprises the extra electric insulation thin slice (31) of have rear surface (31b) and hydrophobic front surface (31a), described extra electric insulation thin slice (31) is removably connected on the described front surface (27 ') of described the second substrate (27), (31b) is adhered to described front surface (27 ') in wherein said rear surface, described extra electric insulation thin slice (31) has one or more reagent storage bin (12), described one or more reagent storage bin (12) is positioned on one or more pre-selected position (13) of described hydrophobic front surface (31a) of described extra electric insulation thin slice (31).

21. digital micro-fluid devices as claimed in claim 20 (14), it is characterized in that: described device comprises the upper extra electrod-array (35) of described front surface (27 ') that is arranged on described the second substrate (27), and the described extra electric insulation thin slice (31) that described extra electrod-array (35) is had hydrophobic front surface (31a) covers.

22. digital micro-fluid devices as claimed in claim 21 (14), is characterized in that: described device comprises the dielectric layer (25) being clipped between described extra electric insulation thin slice (31) and the described front surface (27 ') of described extra electrod-array (35) and described the second substrate (27).

23. 1 kinds of digital micro-fluid methods, said method comprising the steps of:

(a) prepare digital micro-fluid device (14), described digital micro-fluid device (14) is included in the first substrate (24) and above arranges by dispersive electrode (17) electrod-array (16) forming; And electrode controller (19), described electrode controller is arranged with described dispersive electrode (17) the described electrod-array (16) forming and is connected, for applying selected voltage pattern optionally to start and to stop described dispersive electrode (17) to described dispersive electrode (17), thereby liquid sample drop (20,22) is above passed through to described electrod-array (16) with desirable approach at described dispersive electrode (17);

(b) provide prepackage carrier (10), described prepackage carrier (10) has electric insulation thin slice (11), described electric insulation thin slice (11) has hydrophobic front working surface (11a) and rear surface (11b), described electric insulation thin slice (11) has one or more reagent storage bin (12), and described one or more reagent storage bin (12) are located in the one or more pre-selected position (13) on the described front working surface (11a) of described electric insulation thin slice (11);

(c) the described rear surface (11b) of described electric insulation thin slice (11) is connected on the surface (16 ') of described electrod-array (16) of described digital micro-fluid device (14), in the time being placed on described electrod-array (16), described electric insulation thin slice (11) thus cover described dispersive electrode (17), and make described dispersive electrode (17) electrically insulated from one another and with drop (20 on described hydrophobic front surface (11a), 22, 33) electric insulation, and the described one or more pre-selected position (13) on the described front working surface (11a) of described electric insulation thin slice (11) is arranged to approach the upper drop starting of described front working surface (11a) of described electric insulation thin slice (11),

(d) test, guide one or more sample droplets (20,22) by extremely described one or more reagent storage bin (12) of described front working surface (11a), by this by described one or more sample droplets (20,22) be delivered to by described one or more sample droplets (20,22) described one or more reagent storage bin (12) of reformulating, and at least one the selected reagent mix comprising with described one or more reagent storage bin (12);

(e) product (26) forming between described at least one the selected reagent at least one of described one or more reagent storage bin (12) and described sample droplets of mixing (20,22) and obtain is separated; And

(f) the electric insulation thin slice (11) of peeling described connection off from the described surface (16 ') of the described electrod-array (16) of described digital micro-fluid device (14), to remove the electric insulation thin slice (11) of described connection, can be reused described electrod-array (16) thereby go up new prepackage carrier (10) by connection.

24. digital micro-fluid methods as claimed in claim 23, it is characterized in that: the described surface (16 ') that described electric insulation thin slice (11) is connected to described electrod-array (16) by adhesive (15) is upper, and wherein said adhesive (15) contacts the surface (24 ') of the described rear surface (11b) of described electric insulation thin slice (11) and the described surface (16 ') of described electrod-array (16) and/or described the first substrate (24).

25. digital micro-fluid methods as described in claim 23 or 24, is characterized in that: described rear surface (11b) is connected on the described surface (16 ') of described electrod-array (16).

26. digital micro-fluid methods as described in claim 23 or 24, is characterized in that: described method comprises the step (g) of the product (26) obtaining described in analysis.

27. digital micro-fluid methods as claimed in claim 26, is characterized in that: the step (g) of the described product of described analysis (26) was carried out before or after the electric insulation thin slice (11) of removing described connection according to step (f).

28. as claim 23, digital micro-fluid method in 24 or 27 described in any one, it is characterized in that: the one or more sample droplets (20 of described guiding, 22) comprise from one or more sample container (32) and distribute described one or more drop (20 by the step (d) of described front working surface (11a), 22, 33), described one or more sample container (32) is arranged on to approach and is positioned at the described front working surface (11a) of being arranged the described electric insulation thin slice (11) on the described electrod-array (16) forming by described dispersive electrode (17).

29. digital micro-fluid methods as described in any one in claim 23,24 or 27, is characterized in that: described one or more reagent storage bin (12) comprise the biological substrate for cell adhesion.

30. digital micro-fluid methods as described in any one in claim 23,24 or 27, it is characterized in that: in step (d) by described one or more sample droplets (20,22) be exposed to after described at least one selected reagent storage bin (12), by each sample droplets (20,22) pass through described dispersive electrode (17) with the mixture translation of described at least one selected reagent, and merge and mix with one or more other sample droplets (20,22).

31. digital micro-fluid methods as described in any one in claim 23,24 or 27, it is characterized in that: in step (d) by described one or more sample droplets (20,22) be exposed to after described at least one selected reagent storage bin (12), by each sample droplets (20,22) pass through described dispersive electrode (17) with the mixture translation of described at least one selected reagent, and be exposed at least one selected reagent storage bin (12).

32. digital micro-fluid methods as described in any one in claim 23,24 or 27, it is characterized in that: in step (d) by described one or more sample droplets (20,22) be exposed to after described at least one selected reagent storage bin (12), by each sample droplets (20,22) be divided into one or more appended sample drops with the mixture of described at least one selected reagent, and one or more appended sample drops described in processing, Collection and analysis.

33. digital micro-fluid methods as described in any one in claim 23,24 or 27, it is characterized in that: step (d) comprise from described working surface (11a) keep flowing one or more solvent containers (34) of connected state one or more drops (33) of one or more solvent are guided to described one or more selected dispersive electrodes (17), to dissolve before described one or more reagent at the described one or more sample droplets of guiding (20,22) to described one or more selected dispersive electrodes (17).

34. digital micro-fluid methods as claimed in claim 29, is characterized in that: described biological substrate comprises in fibronectin, collagen, laminin, polylysin any and combination thereof.

The kit of 35. 1 kinds of enforcement digital micro-fluid method as described in any one in claim 23 to 34, it is characterized in that: described kit comprises the carrier (10) of the prepackage reagent storage bin (12) as described in any one in claim 1 to 8, described carrier (10) has electric insulation thin slice (11), described electric insulation thin slice (11) has hydrophobic front surface (11a) and rear surface (11b), be connected in digital micro-fluid device (14) before described reagent storage bin (12) be arranged on the hydrophobic front surface (11a) of described electric insulation thin slice (11).

36. kits as claimed in claim 35, is characterized in that: described kit comprises the digital micro-fluid device (14) as described in any one in claim 9 to 22.

37. kits as described in claim 35 or 36, is characterized in that: described prepackage carrier (10) is packaged in a packaging with multiple other carriers (10).

38. kits as claimed in claim 37, it is characterized in that: be packaged in the described prepackage carrier (10) in a packaging every kind of carrier and have the reagent storage bin (12) of equal number, wherein each reservoir (12) comprises identical reagent composition.